Cyclodextrins are cyclic oligosaccharides. The most common cyclodextrins are .alpha.-cyclodextrin, which is composed of a ring of six glucose residues; .beta.-cyclodextrin, which is composed of a ring of seven glucose residues; and .gamma.-cyclodextrin, which is composed of a ring of eight glucose units. The inside cavity of a cyclodextrin is lipophilic, while the outside of the cyclodextrin is hydrophilic; this combination of properties has led to widespread study of the natural cyclodextrins, particularly in connection with pharmaceuticals, and many inclusion complexes have been reported. .beta.-Cyclodextrin has been of special interest because of its cavity size, but its relatively low aqueous solubility (about 1.8% w/v at 25.degree. C.) and attendant nephrotoxicity have limited its use in the pharmaceutical field.
Attempts to modify the properties of the natural cyclodextrins have resulted in the development of heptakis (2,6-di-O-methyl)-.beta.-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-.beta.-cyclodextrin, hydroxypropyl-.beta.-cyclodextrin, .beta.-cyclodextrin-epichlorohydrin polymer and others. For a comprehensive review of cyclodextrins and their use in pharmaceutical research, see Pitha et al, in Controlled Drug Delivery, ed. S. D. Bruck, Vol. I, CRC Press, Boca Raton, Fla., pp. 125-148 (1983). For an even more recent overview, see Uekama et al, in CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1), 1-40 (1987); Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. D. Breimer and P. Speiser, Elsevier Science Publishers B. V. (Biomedical Division), 181-194 (1987); and Pagington, Chemistry in Britain, pp. 455-458 (May 1987).
Inclusion complexes of .alpha.-, .beta.- or .gamma.-cyclodextrin or their mixtures with a variety of drugs have been described by numerous parties and various advantages have been attributed to the complexes. These descriptions include the following:
__________________________________________________________________________ U.S. ACTIVE INVENTOR PAT. NO. INGREDIENT USE ADVANTAGE __________________________________________________________________________ Noda et al 4,024,223 menthol &/or antiphlogistic, reduced unpleasant methyl analgesic odor, increased salicylate wet packing effect Szejili et al 4,228,160 indomethacin anti-inflam- reduced ulcerative matory, pro- effect tective during pregnancy Hayashi et al 4,232,009 .omega.-halo-PGI.sub.2 hypotensive, increased stability analogs uterine con- traction stimulating, blood platelet aggregation inhibiting Matsumoto et al 4,351,846 3-hydroxy-and uterine contrac- increased stability 3-oxo- tion stimulating prostaglandin analogs Yamahira et al 4,352,793 bencyclane anticonvulsant, increased stability fumarate vasodilative at strong acid pH, faster gastric emptying, higher blood concentrations, less irritation, improved hemolytic activity Lipari 4,383,992 steroids- hormonal improved water corticosteroids, solubility, increased androgens, therapeutic response anabolic in eye steroids, estrogens, progestagens Nicolau 4,407,795 p-hexadecyl- antiathero- enhanced aminobenzoic sclerotic bioavailability acid sodium salt Tuttle.sup.1 4,424,209 3,4-diisobutyr- cardiac yloxy-N-[3-(4- contractility isobutyryloxy- agent phenyl)-1- methyl-.alpha.- propyl]-.beta.- phenethylamine Tuttle 4,425,336 3,4-dihydroxy- cardiac capable of oral N-[3-(4-hydroxy- contractility administration phenyl)-1- agent methyl-n- propyl]-.beta.- phenethylamine Wagu et al 4,438,106 EPA and DHA deodorized, (fatty acids) storage stable Masuda et al.sup.2 4,474,811 2-(2-fluoro-4- anti- reduced eye biphenylyt)pro- inflammatory irritation, pionic acid ophthalmic higher concen- or salt trations, no side effects, highly soluble, long stability, excellent pharmacological effects Shinoda et al 4,478,995 acid addition anti-ulcer excellent water salt of (2'- solubility, good benzyloxycar- absorption in diges- bonyl)phenyl tive tract, good trans-4-guani- anti-ulcer activity dinomethylcyclo- hexanecarboxylate Hayashi et al 4,479,944 PGI.sub.2 analog for treatment of stabalization against artereosclerosis, decomposition cardiac failure or thrombosis Hayashi et al 4,479,966 6,9-methano- for hypertension, increased stability PGI.sub.2 analogs cerebral throm- bosis and the like Harada et al 4,497,803 lankacidin- antibiotic for enhanced water group antibiotic swine dysentery solubility and stability, increased rate and amount of absorption Masuda 4,499,085 prostaglandin treating anoxia analog of brain cells Szejtli et al 4,518,588 phendiline, i.e. coronary dilator improved water sol- N-(1-phenyl- calcium ubility, accelerated ethyl)-3,- antagonist and increased in diphenylpro- vivo resorption pylamine or its & dissolution at pH/ hydrochloride temperature of gastric acid Szejtli et al 4,524,068 piperonyl synergizes easily handled butoxide pesticidal effect crystalline solid; of known insecti- improved water sol- cides and fungi- ubility, increased cides absorption & velocity of penetration through biological membranes Jones 4,555,504 a cardiac cardiac effect high aqueous solu- glycoside bility, apparently better bioavail- ability Uekama et al.sup.3 4,565,807 pirprofen anti-inflam- improved stability matory, to oxidation, analgesic, freedom from bitter antipyretic taste, less irrita- ting Ueda et al 4,575,548 2-nitroxymethyl- for vascular non-volatile powder 6-chloropyridine disorders vs. volative oil Ohwaki et al.sup.4 4,598,070 tripamide anti-hyper- improved solubility tensive Chiesi et al 4,603,123 piroxicam, i.e. anti-inflam- 4-hydroxy-2- matory, analgesic methyl-N-2- pyridyl-2H-1,2- benzothiazine-3- carboxamide-1,1- dioxide Hasegawa et al 4,608,366 mobenzoxamine, antiemetic, storage stability, i.e. 1-[2-(4- antispasmodic better absorption methoxybenzhy- through digestive dryloxy)ethyl]- tract 4-(3-(4-fluoro- benzoyl)propyl]- piperazine Hirai et al.sup.2 4,659,696 polypeptide improving drug absorption by non- oral and non- injection routes Szejtli et al 4,623,641 PGI.sub.2 methyl anti-ulcer improved storage ester stability Ninger et al 4,663,316 unsaturated antibiotic, enhanced stability phosphorus- antifungal, against oxidation containing antitumor antibiotics, including phosphotrienin Fukazawa et al 4,675,395 hinokitiol bactericidal, improved water sol- bacteriostatic ubility, less odor Shimizu et al 4,728,509 2-amino-7- anti-allergic, improved water sol- isopropyl-5- anti- ubility to increase oxo-5H-[1]- inflammatory concentration to benzopyrano- therapeutic levels [2,3-b]pyridine- in nasal drops and 3-carboxylic acid eye drops Shibanai 4,728,510 a milk bath improved stability et al.sup.6 component preparation Karl et al 4,751,095 aspartame dipeptide stabilization from sweetener hydrolysis __________________________________________________________________________ .sup.1 Tuttle also describes use of 2,6di-O-methyl-cyclodextrin to form the inclusion complex. .sup.2 This may not be an inclusion complex, but simply a physical mixture. .sup.3 This is a mixture and/or an inclusion compound. .sup.4 The inventors also mention prior known solubility improvements of cyclodextrin inclusions of barbituric acid derivatives, mefenamic acid, indomethacin and chloramphenicol. .sup.5 The inventors refer to this as an "occlusion" compound. .sup.6 The inventors also mention a derivative of cyclodextrin and a cyclodextrincontaining starch decomposition product for use in forming th clathrate.
Among the patents listed above, only Hirai et al U.S. Pat. No. 4,659,696 appears to relate to proteins or polypeptides. It would appear from the claims of the Hirai et al patent, however, that an inclusion complex is not formed and that what the invention provides is a pharmaceutical composition consisting essentially of a physical mixture of a hydrophilic, physiologically active polypeptide and cyclodextrin, said composition being a uniform mixture in dosage form. The cyclodextrin may be .alpha.-, .beta.- or .gamma.-cyclodextrin, or a cyclodextrin derivative, exemplified by tri-O-methylcyclodextrin and triaminocyclodextrin, .alpha.-cyclodextrin being particularly preferred. The physiologically active peptide may be thyrotropin releasing hormone, luteinizing hormone-releasing hormone, insulin, somatostatin, growth hormone, prolactin, adrenocorticotrophic hormone, melanocyte stimulating hormone, or many others. The composition is designed for nasal, vaginal or rectal preparation, i.e. a non-oral or non-injectable route of administration is used.
Matsuyama et al, Drug Development and Industrial Pharmacy, 13(15), 2687-2691 (1987), have reported on the thermodynamics of binding aromatic amino acids to .alpha.-, .beta.- and .gamma.-cyclodextrin. Human serum albumin, which contains aromatic amino acids, was also tested as a protein model. The authors suggest that cyclodextrins may protect peptides from enzymatic hydrolysis. Also, according to Sekisui Chemical Co., Ltd.'s Japanese Kokai Tokkyo Koho JP 59/104556 (84/104556), published Jun. 16, 1984, cyclized oligosaccharides such as .beta.-cyclodextrin have been recently used in protein stabilizers which prevent denaturation of proteins and enzymes in blood. Human blood mixed with octylphenoxypoly(ethoxyethanol) and .beta.-cyclodextrin was maintained for 24 hours with little change.
Inclusion complexes of 2,6-di-O-methyl-.beta.-cyclodextrin with dibenzo[bd]pyran derivatives and salts having analgesic, antiemetic and narcosis-potentiating activities have been described in Nogradi et al U.S. Pat. No. 4,599,327; increased water solubility and thus improved biological activity have been claimed for the complexes. A review of the pharmaceutical applications of such methylated cyclodextrins has been published by Uekama, Pharm. Int., March 1985, 61-65; see also Pitha, Journal of Inclusion Phenomena 2, 477-485 (1984).
Cyclodextrin polymer has been reported by Fenyvesi et al, Chem. Pharm. Bull. 32 (2), 665-669 (1984) to improve the dissolution of furosemide. Improvements in the dissolution and absorption of phenytoin using a water-soluble .beta.-cyclodextrin epichlorohydrin polymer have been described by Uekama et al, International Journal of Pharmaceutics, 23, 35-42 (1985).
Hydroxypropyl-.beta.-cyclodextrin (HPBCD or HPCD) and its preparation by propylene oxide addition to .beta.-cyclodextrin were described in Gramera et al U.S. Pat. No. 3,459,731 nearly 20 years ago. Gramera et al also described the analogous preparation of hydroxyethyl-.beta.-cyclodextrin by ethylene oxide reaction with .beta.-cyclodextrin. Much more recently, Pitha and co-workers have described the improved preparation of this cyclodextrin derivative and its effects on the dissolution of various drug molecules. Pitha U.S. Pat. No. 4,596,795, dated Jun. 24, 1986, describes inclusion complexes of sex hormones, particularly testosterone, progesterone, and estradiol, with specific cyclodextrins, preferably hydroxypropyl-.beta.-cyclodextrin and poly-.beta.-cyclodextrin. The complexes enable the sex hormones to be successfully delivered to the systemic circulation via the sublingual or buccal route; the effectiveness of this delivery is believed to be due to "the high dissolution power of hydrophilic derivatives of cyclodextrins, the non-aggregated structure of their complexes with steroids, and their low toxicity and irritancy of mouth tissue". Success with other cyclodextrins, including poly-.gamma.-cyclodextrin and hydroxypropyl-.gamma.-cyclodextrin, have also been noted in the Pitha patent. See also Pitha et al, J. Pharm. Sci., Vol. 74, No. 9, 987-990 (September 1985), concerning the same and related studies. Pitha et al also describe in the J. Pharm. Sci. article the storage stability of tablets containing a testosterone-hydroxypropyl-.beta.-cyclodextrin complex and the lack of toxicity of the cyclodextrin itself, as well as the importance of the amorphous nature of the cyclodextrin derivatives and their complexes with drugs in improving dissolution properties.
The improved, optimized preparation and purification of hydroxypropyl-.beta.-cyclodextrin have been recently described by Pitha et al, International Journal of Pharmaceutics 29, 73-82 (1986). In the same publication, the authors have described increased water solubility for 32 drugs in concentrated (40 to 50%) aqueous solutions of hydroxypropyl-.beta.-cyclodextrin; improved solubilization of acetaminophen, apomorphine, butylated hydroxytoluene, chlorthalidone, cholecalciferol, dexamethasone, dicumarol, digoxin, diphenylhydantoin, estradiol, estriol, ethinylestradiol-3-methyl ether, ethisterone, furosemide, hydroflumethiazide, indomethacin, iproniazid phosphate, 17-methyltestosterone, nitroglycerin, norethindrone, ouabain, oxprenolol, progesterone, retinal, retinoic acid (all trans and salt forms), retinol, spironolactone, sulpiride, testosterone and theophylline was noted. The authors indicated this to be an extension of their earlier work with hydroxypropyl-.beta.-cyclodextrin, which was previously found effective for oral administration of the sex hormones to humans. Their later work reported in Pitha et al, International Journal of Pharmaceutics 29, 73-82 (1986), has also been very recently described in Pitha U.S. Pat. No. 4,727,064, dated Feb. 23, 1988. That patent claims a composition containing an amorphous complex of cyclodextrin and a drug, and a method of producing a stabilizing amorphous complex of a drug and a mixture of cyclodextrins comprising (1) dissolving an intrinsically amorphous mixture of cyclodextrin derivatives which are water soluble and capable of forming inclusion complexes with drugs in water; and (2) solubilizing lipophilic drugs into aqueous media to form a solution and form a solubilized drug/cyclodextrin complex. Work with numerous drugs has been reported in the patent, but none involves proteins or other peptides. The patent describes the preparation of various substituted amorphous cyclodextrins, including hydroxypropyl-.beta.-cyclodextrin and hydroxypropyl-.gamma.-cyclodextrin, the latter by analogous condensation of propylene oxide with .gamma.-cyclodextrin.
Uekama et al, CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1), pp. 140 (1987), have described the characteristics of various cyclodextrins, including hydroxypropyl-.beta.-cyclodextrin. The authors have presented data showing improved solubilization in water in the presence of 15 mg/mL of HPBCD for the drugs carmofur, diazepam, digitoxin, digoxin, flurbiprofen, indomethacin, isosorbide dinitrate, phenytoin, prednisolone, progesterone and testosterone. In a discussion of the metabolism and toxicity of cyclodextrins, Uekama et al have indicated that cyclodextrins at low-moderate concentrations (usually 3-8% w/v and higher) cause hemolysis, and that the methylated cyclodextrins have higher hemolytic activity than the natural cyclodextrins. Hydroxypropyl-.beta.-cyclodextrin is said to cause hemolysis beginning at 4.5 mM. The authors have further indicated that parenteral administration of large doses of cyclodextrins should be avoided, but that ".gamma.-cyclodextrin and hydroxypropyl-.beta.-cyclodextrin seem to be useful in drug solubilization for injections and liquid preparations used for mucous membranes." Use with polypeptides such as proteins is not mentioned.
JANSSEN PHARMACEUTICA N.V.'s International Patent Application No. PCT/EP84/00417, published under International Publication No. WO85/02767 on Jul. 4, 1985, has described pharmaceutical compositions comprising inclusion compounds of drugs, which are unstable or only sparingly soluble in water, with partially etherified .beta.-cyclodextrin derivatives having hydroxyalkyl and optionally additional alkyl groups. Among the cyclodextrin derivatives contemplated are hydroxypropyl-.beta.-cyclodextrin and hydroxyethyl-.beta.-cyclodextrin, while the drugs include non-steroidal anti-rheumatic agents, steroids, cardiac glycosides and derivatives of benzodiazepine, benzimidazole, piperidine, piperazine, imidazole and triazole. Preferred drugs include etomidate, ketoconazole, tubulazole, itraconazole, levocabastine and flunarizine. The pharmaceutical compositions of the invention include oral, parenteral and topical formulations, with 4 to 10% solutions of cyclodextrin derivatives being utilized to solubilize various drugs. Improved solubilities of indomethacin, digitoxin, progesterone, dexamethasone, hydrocortisone and diazepam using 10% HPBCD are shown, and an injectable formulation of diazepam in 7% HPBCD is specifically described. The relatively low cyclodextrin concentrations used reflect a desire to avoid or minimize the hemolytic effects observed at higher cyclodextrin concentrations.
Carpenter et al, The Journal of Pediatrics, 111, 507-512 (October 1987) describe intravenous infusion of 2-hydroxypropyl-.beta.-cyclodextrin, prepared as a 5% solution in water, to treat severe hypervitaminosis A. It was found that, during infusion, circulating retinyl esters increased transiently, while total vitamin A excreted in the urine was enhanced after infusion. Thus, intravenous infusion of 5% HPBCD was found to decrease in vivo levels of the vitamin, presumably by complexing with the vitamin and removing some of the excess from the body.
Formulation of a particular dihydropyridine pyridinium salt redox system for brain-specific delivery of estradiol "in a water-soluble .beta.-hydroxy-cyclodextrin" is reported by Bodor and co-workers in Estes et al, "Use of a Chemical Redox System for Brain Enhanced Delivery of Estradiol Decreases Prostate Weight," in Biological Approaches to the Controlled Delivery of Drugs., ed. R. L. Juliano, Annals of the New York Academy of Sciences, Volume 507, 1987, 334-336.
JANSSEN PHARMACEUTICA N.V.'s European Patent Application No. 86200334.0, published under EPO Publication No. 0197571 on Oct. 15, 1986, describes .gamma.-cyclodextrin derivatives which are .gamma.-cyclodextrin substituted with C.sub.1 -C.sub.6 alkyl, hydroxy C.sub.1 -C.sub.6 alkyl, carboxy C.sub.1 -C.sub.6 alkyl or C.sub.1 -C.sub.6 alkyloxycarbonyl C.sub.1 -C.sub.6 alkyl or mixed ethers thereof. Among the specific derivatives named are hydroxypropyl-.gamma.-cyclodextrin and hydroxyethyl-.gamma.-cyclodextrin. Compositions comprising the cyclodextrin derivatives and a drug are also described. See also corresponding Muller U.S. Pat. No. 4,764,604, dated Aug. 16, 1988.
The inclusion characteristics of yet other derivatized cyclodextrins have also been described in the literature. Studies of branched cyclodextrins which are glucosyl and maltosyl derivatives of .alpha.-, .beta.- and .gamma.-cyclodextrin and their inclusion complexes with drugs have recently been reported. Uekama, in Topics in Pharmaceutical Sciences 1987., eds. D. D. Breimer and P. Speiser, Elsevier Science Publishers B. V. (Biomedical Division), 181-194 (1987), has described the effects on bio-pharmaceutical properties of maltosyl and glucosyl cyclodextrin derivatives, including enhanced drug absorption. In that publication, Uekama again indicated that the natural cyclodextrins caused hemolysis in the order of .gamma.&lt;.alpha.&lt;.beta.. In the case of chemically modified cyclodextrins, the order changed to hydroxyethyl-.beta.&lt;maltosyl-.beta.&lt;hydroxypropyl-.beta.&lt;.beta.&lt;methyl-.be ta..
Koizumi et al, Chem. Pharm. Bull. 35 (8), 3413-3418 (1987), have reported on inclusion complexes of poorly water-soluble drugs with glucosyl cyclodextrins, namely 6-O-.alpha.-D-glucosyl-.beta.-CD (G.sub.1 -.alpha.-CD), 6-O-.alpha.-D-glucosyl-.beta.-CD (G.sub.1 -.beta.-CD) and 6A, 6.sup.D -di-O-.alpha.-D-glucosyl-.beta.-CD (2G.sub.1 -.beta.-CD).
Okada et al, Chem. Pharm. Bull. 36 (6), 2176-2185 (1988), have reported on the inclusion complexes of poorly water-soluble drugs with maltosyl cyclodextrins, namely 6-O-.alpha.-maltosyl-.alpha.-CD (G.sub.2 -.alpha.-CD), 6-O-.alpha.-maltosyl-.beta.-CD (G.sub.2 -.beta.-CD), 6-O-.alpha.-maltosyl-.gamma.-CD (G.sub.2 -.gamma.-CD), 6-O-.alpha.-maltotriosyl-.alpha.-CD (G.sub.3 -.alpha.-CD), 6-O-.alpha.-maltotriosyl-.beta.-CD (G.sub.3 -.beta.-CD) and 6-O-.alpha.-maltotriosyl-.gamma.-CD (G.sub.3 -.gamma.-CD).
Yamamoto et al, in International Journal of Pharmaceutics 49, 163-171 (1989), have described physicochemical properties of branched .beta.-cyclodextrins such as glucosyl-.beta.-cyclodextrin, maltosyl-.beta.-cyclodextrin and di-maltosyl-.beta.-cyclodextrin, and their inclusion characteristics. Those authors report that the branched .beta.-cyclodextrins are better solubilizers for poorly water-soluble drugs and have less hemolytic activity than .beta.-cyclodextrin itself, and they suggest that glucosyl-.beta.-cyclodextrin and maltosyl-.beta.-cyclodextrin may be especially useful in parenteral preparations.
The patent literature reflects much recent work on the branched cyclodextrins carried out by Japanese workers, as discussed below.
Japanese Kokai 63-135402 (TOKUYAMA SODA KK), published Jun. 7, 1988, describes compositions consisting of maltosyl-.beta.-cyclodextrin and at least one of digitoxin, nifedipine, flulubiprophene, isosorbide nitrate, phenytoin, progesterone or testosterone. The compositions have improved water solubility and reduced erythrocyte destruction, are safe for humans and can be used as injections, eye drops, syrups, and for topical and mucous membrane application.
Japanese Kokai 62-281855 (DAIKIN KOGYO KK), published Dec. 7, 1987, describes stable, water-soluble inclusion compounds of maltosyl-.beta.-cyclodextrin with a variety of vitamins and hormones, e.g. steroid hormones such as prednisolone, hydrocortisone and estriol. These lipophilic vitamins and hormones can thus be used as aqueous solutions.
Japanese Kokai 63-036793 (NIKKEN CHEM KK), published Feb. 17, 1988, describes the preparation of dimaltosyl-.gamma.-cyclodextrin and its general use in medicines.
Japanese Kokai 62-106901 (NIKKEN CHEM KK), published May 18, 1987, describes the preparation of diglucosyl-.beta.-cyclodextrin and its general use for pharmaceuticals.
Japanese Kokai 61-236802 (NIKKEN CHEM KK), published Oct. 22, 1986, describes the preparation of maltosyl-.gamma.-cyclodextrin and its general use with drugs.
Japanese Kokai 61-197602 (NIKKEN CHEM KK), published Sep. 1, 1986, describes the preparation of maltosyl-.beta.-cyclodextrin and its expected use in medicines.
Japanese Kokai 61-070996 (NIKKEN CHEM KK), published Apr. 11, 1986, describes the preparation of maltosyl-.alpha.-cyclodextrin and its general use in pharmaceuticals.
Japanese Kokai 63-027440 (SANRAKU OCEAN), published Feb. 5, 1988, describes compositions containing a water-insoluble or slightly soluble drug together with glucosylated branched cyclodextrin. Among the drugs mentioned are steroid hormones.
Japanese Kokai 62-164701 (SHOKUHIN SANGYO BIO), published Jul. 21, 1987, describes the preparation of diglucosyl-.alpha.-cyclodextrin and its general use in medicine.
Japanese Kokai 62-003795 (TOKUYAMA SODA KK), published Jan. 9, 1987, describes production of glucose and maltoligosaccharide (2-4 glucose units) derivatives of .alpha.-, .beta.- and .gamma.-cyclodextrin and their use as stabilizers for pharmaceuticals.
In recent years, there has been a tremendous increase in the number of products and potential products containing polypeptides, especially proteins, intended for therapeutic or diagnostic or analytical use. Typically, the proteins or other peptides are formulated in aqueous solution for injection or for use in diagnostic or analytical applications. Unfortunately, proteins often suffer from solubility and/or stability problems. Some proteins, for example, are not water-soluble. Others are soluble in water but still suffer from stability problems caused by protein degradation/denaturation, dimerization and/or polymerization, and the like, any of which may lead to inactivation. This seriously limits shelf-life and often imposes low-temperature storage requirements and restrictions on mechanical movement.
There are numerous causes of protein/peptide instability or degradation, including covalent bond reactions, such as hydrolysis, and the denaturation process. In the case of denaturation, the conformation or three-dimensional structure of the protein is disturbed and the protein unfolds from its usual globular structure. Rather than refolding to its natural conformation, hydrophobic interaction may cause clumping of molecules together, or aggregation, or it may cause refolding to an unnatural conformation. Either of these end results entails diminution or loss of biological activity. For a summary of the causes of protein instability, see Wang et al, Journal of Parenteral Science and Technology, Technical Report No. 10, "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", Supplement Volume 42, Number 25, 1988, pp. S3-S26.
The Wang et al publication referenced above also summarizes the art's use of various excipients to stabilize parenteral formulations of proteins and other peptides, including use of serum albumin, amino acids, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinylpyrrolidone, hydrolyzed gelatin and ammonium sulfate, including agents used as cryoprotectants, which prevent or lessen denaturation under freezing conditions or lyophilization, such as carbohydrates, alcohols, polyvinylpyrrolidone and glutamic acid. Examples of excipients proposed by the art for stabilizing specific proteins are described in Hayashi et al U.S. Pat. No. 4,457,916, issued Jul. 3, 1984, KYOWA HAKKO KOGYO CO, LTD.'s European Patent Publication No. 0123291 and E. I. DUPONT DE NEMOURS AND COMPANY'S European Patent Publication No. 0231132.
The Hayashi et al patent describes a method for stabilizing Tumor Necrosis Factor (TNF) by adding a stabilizing agent selected from a nonionic surfactant, at least one substance selected from D-glucose, D-galactose, D-xylose, D-glucuronic acid, a salt of D-glucuronic acid, trehalose, a dextran and a hydroxyethyl starch and mixtures thereof. The resultant aqueous solution or powder containing TNF is said to be capable of prolonged storage without loss of activity and stable on freezing, thawing, lyophilization, heat-treatment or the like.
European Patent Publication No. 0123291, published Oct. 31, 1984, describes a method for stabilizing interferon involving addition of an alkali metal salt or magnesium chloride to interferon, optionally including addition of serum albumin and freeze-drying to further enhance stability. The patent publication also describes a method for stabilizing interferon by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulfuric acid, starch, glycogen, insulin, dextrin and alginic acid salt.
European Patent Publication No. 0231132, published Aug. 5, 1987, relates to a recombinant interleukin-2 (IL-2) composition and process for making it. In discussing the prior art, the European publication refers to earlier European Patent Publication No. 0133767 as disclosing that gamma-interferon, obtainable from human leukocytes, can be stabilized by addition of albumin and/or a sugar. The sugars can include monosaccharides, disaccharides, sugar alcohols and mixtures thereof, e.g. glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol and xylitol. Also, European Patent Publication No. 0231132 acknowledges that protein (chymotrypsinogen and ribonuclease) stabilization using glycerol is reported by Gekko et al, Biochemistry 20, 4677-4686 (1981). European No. 0231132 itself relates to recombinant human IL-2 compositions comprising water, recombinant interleukin-2 and preferably a polyol. Suitable polyols are said to be water-miscible or water-soluble, for example polyhydroxy alcohols, monosaccharides and disaccharides including mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, glucose, fructose, arabinose, mannose, maltose and sucrose. The polyol is said to increase the activity of the IL-2.
Despite the foregoing, there remains a clear and pressing need in this art for a more effective and generally applicable method and compositions for the solubilization and stabilization of polypeptides such as proteins.